DESCRIPTION: Particle elasticity is a pivotal influence to cellular transport and thereby essential components to nanomedicine design; however, nanoparticle mechanics remain under-characterized, limited in scope by technology, leaving this critical design space untapped and misunderstood. Left unattended, this critical need continues to obscure the expedient development of next- generation nanotherapeutics. The long-term goal of this research program is to determine the key nano-mechanical properties that enable particle discrimination at the cell membrane for deliberate endocytic selection. The objective of this application is to co-polymerize a model hydrogel (N-isopropylacrylamide, NIPAM) with polystyrene (PS) for tailoring a nanoparticle library of discrete size and elasticity for subsequent in vitro discrimination of thir relative influence on cellular uptake efficiency and endocytic selection. Principal to this application, micro- Brillouin light scattering (-BLS) is offered as uniquely well-suited for the comprehensive characterization of nanoparticle mechanics beyond current capabilities. Our central hypothesis follows that cellular selection of endocytic mechanism arises from independent recognition of particle size and mechanics, i.e. particles that are size-forbidden can be permitted entry through an alternative mechanosensitive mechanism. Our rationale for this hypothesis is based on our preliminary data indicating clear adjustment of identically sized PS (stiff) and PS-co-NIPAM (soft) particles into a macrophage cell line. It is emphasized that our approach is the first of its kind for isolating particle size and mechanics to clearly demonstrate their relative endocytic influence. To accomplish our central hypothesis, our first aim is to establish -BLS as a comprehensive analytical tool for detailing nanomechanics. Our preliminary BLS spectra indicate sensitive determination of particle elasticity at the nanoscale and moreover verify the inadequacy of assuming bulk and nanoscale mechanics are the same. Our second aim emphasizes discriminating the relative influence of particle size and mechanics for endocytic entry into three model cell lines. Detailing the mechanosensitivity of endocytosis will offer significant contributions to the improved mechanistic development of host recognition in virology, immunology and drug-delivery. Our innovation stems from the extension of BLS and elasticity theory to interpreting cell-nanoparticle dynamics for advanced nanomedicine design. Overall, the novel design strategies that becomes available specifically through this application underscore our alignment with the NIH mission of advancing public health.